Periodontitis is an inflammatory disease of the oral cavity that can lead to bone destruction and tooth loss [1]. It is one of the most prevalent diseases in the world, affecting almost half of all American adults (64.7 million individuals) [2]. Multiple microbes have been associated with this disease, including several Gram-negative opportunistic pathogens [3,4]. It is thought that disease phenotypes are exacerbated by interspecies interactions between pathogens [5] as well as between pathogens and the commensal oral flora [6]. The complexity of this interaction network has hindered our understanding of periodontal disease, and efforts utilizing model polymicrobial communities [6,7] and animal models for oral cavity infection [5,8] have greatly benefitted the field. Previously, we demonstrated that synergistic virulence between the Gram-negative opportunistic pathogen Aggregatibacter actinomycetemcomitans (Aa) and the Gram-positive commensal Streptococcus gordonii (Sg) is mediated by metabolite cross-feeding [9] and by a microbial flight response that results in optimal spacing of Aa from Sg during infection [10]. This flight response is orchestrated by the Aa biofilm-dissolving enzyme Dispersin B (DspB), and we found that DspB is regulated by the iron-sensing regulator Fur. Therefore, the first goal of this proposal is to explore the potential role of this regulation as a strategy by Aa to avoid iron restriction, a majo host immunity factor. The second objective of this proposal is to apply next-generation sequencing technologies [11] to define the Aa transcriptome and genome-wide fitness determinants in two infection environments: (a) during co-infection with Sg in a murine abscess and (b) during infection of the rhesus macaque oral cavity. Our principal hypothesis is that Aa responds to cues from both the commensal flora and the host to establish periodontal infections. The output of these studies will be highly comprehensive, publicly available data sets that will elucidate novel disease mechanisms by the periodontal pathogen Aa. Furthermore, coaggregation has been described among oral microbes for decades [12], suggesting that these microbes actively modulate their physical arrangement as a fitness benefit, and our studies will describe potential mechanisms by which non-motile oral bacteria can control spatial orientation in vivo.